Calmodulin is a small protein involved in the regulation of a wide variety of intracellular processes. The cooperative binding of Ca2+ to calmodulin's two Ca2+ binding domains induces conformational changes which allow calmodulin to activate specific target enzymes. The association of calmodulin with a peptide corresponding to the calmodulin binding site of rabbit smooth muscle myosin light chain kinase (smMLCKp) was studied using isothermal titration microcalorimetry. The dependence of the binding energetics on temperature, pH, Ca2+ concentration, and NaCl concentration were determined. It is found that the binding of calmodulin to smMLCKp proceeds with negative changes in enthalpy ( H), heat capacity ( Cp), and entropy ( S) near room temperature, indicating that it is an enthalpically driven process that is entropically unfavorable. From these results it is concluded that the hydrophobic effect - an entropic effect which favors the removal of nonpolar protein groups from water - is not a major driving force in calmodulin-smMLCKp recognition. Although a large number of nonpolar side chains are buried upon binding these stabilize the complex primarily by forming tightly packed van der Waals interactions with one another. Binding at acidic pH was studied in order to assess the contribution of electrostatic interactions to binding. It is found that moving to acidic pH results in a large decrease in the Gibbs free energy of binding but no change in the enthalpy, indicating that electrostatic interactions contribute only entropically to the binding energetics. The accessible surface area and atomic packing density of the calmodulin-smMLCKp crystal structure are analyzed, and the results discussed in relation to the experimental data.